An organism of the structure mentioned above is known from the document WO 03/064650. This organism is suitable for the biosynthesis of 7-dehydrocholesterol. The motivation for creating this organism is based on the need to produce precursor products to vitamin D3, in particular 7-dehydrocholesterol, in large amounts and under economical conditions.
In a completely different context, there is a need of membrane proteins, in particular human membrane proteins, functionally in a native conformation in at least analytically well acceptable amounts or to make systems available that contain these proteins in a functionally active manner in an amount being for instance sufficient for screening purposes. In principle, for instance biosynthesis methods by means of microorganisms can be used for this. The heterologous expression of membrane proteins from mammal cells in wild type strains, however, often involves difficulties because of the lack of cholesterol, or the presence of ergosterol, respectively, since the expressed membrane proteins are often functionally limited or inactive. This can be explained by the well known fact that sterols in the membranes play an important role for correct folding and the induction of an active conformation of membrane proteins and thus their functionality, i.e. for a functional expression of heterologous membrane proteins in a yeast, the sterol pattern of the membranes must match the membrane protein.
Sterols are an essential constituent of the membranes of eukaryotic cells. They are responsible for the fluidity and permeability of the membranes. Particularly noteworthy is their contribution to the regulation of numerous membrane proteins. They are important as a kind of cofactors for correct folding, stability and activity of the membrane proteins. Free sterols can be found in eukaryotic cells in the plasma membrane and the membranes of all cells compartments. In the lipid particles, sterols occur in an esterified form as storage lipids. In yeasts, the largest part of the free sterols is located in the plasma membrane, followed by the secretory vesicles, and the amount in microsomes, vacuoles and mitochondrial membranes is small. Ergosterol is the final product of the sterol biosynthesis pathway for instance in the yeast S. cerevisiae and is the main sterol in the plasma membrane and the secretory vesicles. The membranes of the subcellular compartments contain smaller amounts of sterols, but also other sterol intermediates.
In mammal cells, the final product of the sterol biosynthesis is cholesterol, which is the largest part of the sterols in the plasma membrane. The plasma membrane contains approx. 60-80% of the total cellular cholesterol, the ER, the place of the sterol synthesis, contains only approx. 0.5-1%.
For instance, in the document Wildt, S., Gerngross, T. U., Nature Reviews (2005)3:119-128, a humanization of yeasts is discussed. The term humanization herein relates, however, to the glycosylation pattern of heterologously produced proteins, which has been adapted to human protein, and not to the sterol pattern being relevant for the present invention. Mainly, the synthesis of enzymes or antibodies is treated, and not the one of membrane proteins.
The ergosterol biosynthesis pathway of yeasts can be subdivided into the pre-squalene pathway, i.e. the synthesis of squalene from acetyl-CoA molecules, and the post-squalene pathway, in which the reaction of squalene to ergosterol is catalyzed. As main pacemaker enzyme of the pre-squalene pathway, the hydroxy-methylglutaryl coenzyme A reductase (HMG-CoA reductase) was identified. The high energy-consuming ergosterol biosynthesis pathway is regulated mainly by this enzyme, which is subject therefore to numerous regulation mechanisms, such as e.g. the feedback inhibition by ergosterol. The pre-squalene and the post-squalene pathway, up to the synthesis of zymosterol, proceed in yeast and mammal cells in the same way. The differences downstream of zymosterol to ergosterol or cholesterol are explained in the following. With yeast cells, zymosterol is reacted by C24 methyltransferase to fecosterol, then by delta7-delta8 isomerase to episterol, by delta5 desaturase to ergosta-5,7,24(28)-trienol, by delta22 desaturase to ergosta-5,7,22,24(28)-tetraenol and finally by delta24 reductase to ergosterol. With mammal cells, zymosterol is reacted by delta7-delta8 isomerase to cholesta-7,24-dienol, by delta5 desaturase to cholesta-5,7,24-trienol and by delta7 reductase to desmosterol, and finally by sterol-delta5 desaturase to cholesterol. The last-mentioned enzyme can also react cholesta-7,24-dienol to lathosterol and cholesta-5,7,24-trienol to 7-dehydrocholesterol. Lathosterol in turn can be reacted by delta5 desaturase to 7-dehydrocholesterol. The latter in turn reacts by delta7 reductase also to cholesterol.
An example for a human membrane protein is the serotonin transporter (SERT). It belongs to the family of the Na+/Cl−-dependent neurotransmitter transporters, which are responsible for the re-uptake of biogenic amines from the synaptic space back to the presynaptic neurons. Further members of this family are the transporters for noradrenaline, dopamine, choline, glycine and γ-aminobutyric acid. The serotonin transporter has a high clinical importance as a pharmacological target molecule of many antidepressants. The antidepressants, the target of which is the SERT, can be subdivided into two main groups: On the one hand the tricyclic agent molecules, such as e.g. imipramine, desimipramine, clomipramine, and on the other hand the SSRI's (selective serotonin re-uptake inhibitors). To the latter group belong active agents such as e.g. paroxetine, fluoxetine, sertraline and citalopram.
Up to now, the SERT was cloned from the human, rat, mouse, bovine and fruit fly Drosophila melanogaster tissue. The SERT was expressed in all common systems, i.e. in E. coli and the yeast Pichia pastoris, in insect cells and in different cell lines, such as for instance COS-1 cells, BHK cells, Hela cells and HEK cells. An expression of the SERT in the yeast Saccharomyces cerevisiae is not known. Up to now, a functional expression could only be achieved in mammal cell lines, and in insect cells, not however in E. coli or Pichia pastoris. The SERT is an extremely difficult protein with regard to heterologous or overexpression. Difficulties occur mainly by the dependence of the serotonin transporter on cholesterol in the membrane surrounding the protein and the importance of the glycosylation for the correct folding of the protein. An activity of the serotonin transporter can only be detected, when cholesterol is present in the membrane. Presumably, this sterol induces a conformal condition of the SERT, which is optimal for its activity. Up to now, it was assumed that the lack of cholesterol in the membrane cannot be compensated by substitution by other sterols. Because of the importance of the cholesterol for the SERT, it is clear, why a functional expression in E. coli has not succeeded up to now. By western blot experiments, Tate et al. (Tate, C. G., Haase, J., Baker, C., Boorsma, M., Magnani, F., Vallis, Y., and Williams, D. C., Biochimica et Biophysica Acta (2003) 1610, 141-153) could show that the rSERT is glycosylated, but not functionally expressed in Pichia pastoris. The assumption is obvious that the lack of cholesterol in the yeast is responsible for this.
The documents Xu S H, Nes W D, Biochem Biophys Res Commun. (1988)155(1):509-17 and WO-2005/121315 A describe cholesterol-producing yeasts. In a different context, a yeast-based system for use in screening methods is known from the document US-2005/0054108 A1.
A modified organism, for instance a strain of the yeast Saccharomyces cerevisiae, which is capable to synthesize cholesterol or a pre-stage, which can compensate the lack of cholesterol with regard to the serotonin transporter, would for the first time give point to making possible a functional expression of this protein in non-mammals. In the case of a functional expression, such an organism could be used as a basis for the design of a “bioassay”. With this “bioassay”, novel pharmacologically relevant active agents for the serotonin transporter could be identified by the “screening” different substance groups. Furthermore, such a yeast strain could also be used for the functional expression of other cholesterol-dependent membrane proteins. The list of the human membrane proteins, for which a cholesterol dependence was detected or is assumed, is long and grows constantly. The list of the diseases and infections, in the pathogenesis of which cholesterol-dependent proteins are involved, is equally long. A system being suitable as a platform for the expression of these proteins could therefore be extremely valuable.